DESCRIPTION: The objective of this study is to understand the molecular basis of Leber's Hereditary Optic Neuropathy (LHON), a maternally inherited disease of young adults which features rapid and painless bilateral loss of central vision due to optic nerve atrophy. LHON results from missense mutations in the mitochondrial DNA (mtDNA) and is genetically heterogeneous as 16 different mtDNA mutations have been associated with the diseases. Five mutations are considered high risk (primary) etiological factors and together account for roughly 805 of all LHON patients in the US and Europe. All high-risk LHON mutations are found in mtDNA encoded subunits of respiratory Complex I (NADH) dehydrogenase), suggestive of a common biochemical defect. However, very little is known about the functional consequences of these mutations, thus the underlying molecular mechanism(s) of this bioenergetic disease remains unsolved which, in turn, has prevented the development of any form of efficacious therapy. To determine the nature of the Complex I defect in LHON patients, two sets of experiments are proposed. First, oxidative phosphorylation biochemical defects will be investigated in patient and control lymphoblastoid cell lines. Both polarographic and spectrophotometric techniques will be used to assay mitochondrial respiration and respiratory enzyme specific activity, respectively. Patient groups to be studied include those harboring only a primary mutation, those harboring only secondary mutations, and those containing both primary and secondary LHON mutations. Second, Paracoccus denitrificans and Escherichia coli will be used as model organisms to study the consequences of the LHON Complex I mutations on enzyme structure and function. Amino acids altered by LHON mutations are conserved in these well- characterized bacteria, in which genetic manipulations are straightforward. P. denitrificans contains a NADH dehydrogenase which is remarkably similar to its mammalian counterpart and E. coli represents a "minimal" proton- translocating NADH dehydrogenase. Primary Complex I LHON mutations will be transferred to the bacteria and mutant bacteria will be assayed for: 1) in vivo aerobic phenotype, 2) specific activity of NADH dehydrogenase, 3) number and function of the critical NADH dehydrogenase Fe-S redox clusters, and 4) structural integrity and stoichiometry of the enzyme complex and its constituent subunits. Completion of this study should result in an understanding of the molecular basis of LHON which will in turn permit the design of effective therapies for this and other bioenergetic diseases.